Scaleable Protein Production in Anchorage Scaleable Protein Production in Anchorage Dependent Mammalian CellsDependent Mammalian Cells
1 Biotechnology Unit, NIDDK,National Institutes of Health, Bethesda, MD 2 Center for Biosystems Research, UMBI, College Park, MD 3 Department of Chemical Engineering, UMCP, College Park, MD
Nicole A. Bleckwenn123, William Bentley23, and Joseph Shiloach1ABSTRACT
Recombinant protein production is a common method for providing clinical and commercial quantities of human therapeutic agents. Methods of production include recombinant bacterial fermentations, transfected or transformed cell culture, and virally infected cell culture. A potential protein production method with anchorage dependent mammalian cells has been evaluated. The system utilizes a recombinant vaccinia virus with the VOTE expression system that can potentially be used to produce large, highly glycosylated recombinant proteins, which may be difficult to produce by other means. A reporter protein, enhanced green fluorescent protein (EGFP), has been used to study the characteristics of this protein production method. Growth, infection and production parameters such as multiplicity of infection, culture volume during infection, infection duration, inducer concentration and timing of inducer addition were studied in monolayer culture. These results were then used to establish conditions for studies in microcarrier spinner flask and 1.5 L bioreactor cultures. Production processes utilizing the selected conditions will be described, together with an evaluation of the expression system.
Objective - Develop an alternative protein production process with recombinant vaccinia virus Necessary components
– Vaccinia Virus Transcription occurs in cytoplasm of infected cell Wide host range VOTE expression system for high, T7 promoter controlled expression
– High cell density HeLa cells – human cervical adenocarcinoma Attachment dependent – growth on microcarriers
Develop bioreactor strategy for growth Study infection process and parameters affecting expression
Project ScopeBACKGROUND
VOTE* Expression System
* Ward, G. A., Stover, C. K., Moss, B., and Fuerst, T. R. (1995). Stringent Chemical and Thermal Regulation of Recombinant Gene- Expression By Vaccinia Virus Vectors in Mammalian-Cells. Proceedings of the National Academy of Sciences of the United States of America 92, 6773-6777.
Recombinant Vaccinia Virus Construction
vT7lacOI
HeLa Cells
Transfect
Infect
vEGFP-His6
PlaquePurify
Insert EGFP-His6
pVOTE.2-EGFP-His6
EGFP-His6
pEGFP-N1
EGFP
pSecTag2A-EGFP
EGFP-His6
InsertEGFP
Infected and Induced
Infected only
Uninfected
Vacuum
LevelControl
FeedPump
WaterJacket
InletOutlet
D.O.
pH
Agit.
Temp.
FiltratePump
AirInlet
MeshScreenModule
Diaphragm
ATFController
Time (hpi)
0 50 100 150 200 250
Via
ble
Ce
ll D
en
sit
y(m
illio
n c
ells
/mL
)
0.0
1.0
2.0
3.0
4.0
5.0
6.0
ATF Feeding Run 1ATF Feeding Run 2Batch Run
Parameters Tested Result
Multiplicity of infection (MOI, pfu/cell) 1.0
Volume during infection Lowest Possible (0.5 mL)
Serum concentration during infection None
Infection duration 1 hr
Inducer concentration 1.0 mM IPTG
Inducer addition timing At time of infection
Time (hpi)
0 10 20 30 40 50 60 70
Intr
acel
lula
r E
GF
P(
g/m
illio
n c
ells
at
infe
ctio
n)
0.0
1.0
2.0
3.0
4.00.10.5 1.0 5.0 10.0 uninfected
Time (hpi)
0 10 20 30 40 50 60 70
Ext
race
llula
r E
GF
P(
g/m
illio
n c
ells
at
infe
ctio
n)
0.0
1.0
2.0
3.0
4.00.10.51.05.010.0uninfected
Time (hpi)
10 20 30 40 50 60 70
EG
FP
(g
/mill
ion
ce
lls a
t in
fec
tio
n)
0.0
2.0
4.0
6.0
8.0
IntracellularExtracellular
Infected Uninfected
Fluo
resc
ence
Ligh
t
FUTURE WORK
MOI Evaluation in Spinner Flasks
RESULTS – Cell Growth
Bioreactor Setup for Microcarrier Culture
ATF Unit Mechanism of ActionA view through the sight glass as the diaphragm moves through an exhaust and pressure cycle
Batch versus ATF Feeding Strategy
6-Well Plate Infection Experiments
RESULTS – Protein Expression
Infection in 1.5 L Bioreactor
lac O EMC SLO
Induced
InactiveRepressor
Target GeneExpression
T7 gene I gpt PL lac IPE/L TT PT7 PE/LTarget
Active Repressor
T7 gene I
lac O
gpt PL lac IPE/L TT
EMC SLO
PT7 PE/LTarget
Uninduced
Cytodex 3 MicrocarriersWith Attached HeLa Cells
More reactor studies–Dissolved oxygen, temperature, feeding strategy
Test production in other cell lines–MRC-5, Vero, HEK 293, etc.
Try other proteins (gp120, hGC-1) Verify post-translational processing
Special Thanks to Bernard Moss and Pat Earl (NIAID, NIH) for providing VOTE componentsRefine Technologies for providing ATF System
Picture of Bioreactor
